Identification Of A Macrolides Antibiotics-producing Strain And Optimization Of Its Fermentation Conditions

In recent years, deep fungal infections increase gradually, which threaten sufferers'health and lives. The discovery and development of efficient, safe, low-toxic and broad-spectrum antifungal drugs become even more important. A strain with high inhibitory effect on Candida albicans ATCC 64548 was screened from East China Sea Soil. Research work on strain identification, fermentation process optimization, antifungal antibiotic structure characterization and antimicrobial efficacy was conducted in this thesis. The strain was identified as Bacillus subtilis based on the taxonomic studies including phenotype determination,16S rRNA gene sequencing and BLAST analysis in Genbank database. It was preserved in China General Microbiological Culture Collection Center, and the identification number was B. subtilis CGMCC 4140.Both culture conditions and medium compositions for producing the antibiotic were optimized by single-factor methodology and response surface methodology (RSM) in 250 mL shaking flask. The culture conditions were optimized as follows: pH 6.5, temperature 28℃, inoculate amount 8%(v/v), shaking speed 200 r/min, liquid volume in flask 40 mL/250 mL, fermentation period 36 h. The optimum composition of culture medium was determined as follows (g/L):soluble starch 27.2, soybean meal 2.77, (NH4)2SO43.73, K2HPO42.0, NaCl 2.0, MgSO41.0, CaCO30.72, CuSO40.02, FeSO40.02, and MnSO40.02. Under the aforementioned culture conditions and medium compositions, the potency activity of culture broth reached to 15766.29 U/mL, which was 15.88-fold compared with the initial conditions.Fermentation scale-up was carried out in 5 L continuous stirred tank reactor (CSTR) with the following culture conditions:rotation speed 300 r/min, broth content 60%(v/v), pH 6.0, inoculum 8%(v/v), aeration rate 1.5 vvm (volume per volume per minute), and fermentation period 28 h. Under the above-mentioned fermentation conditions, the antifungal potency of broth was 1.2-fold compared with which in 250 mL shaking flask. Meanwhile, a mathematical model was developed to describe the kinetics of the macrolide antifungal-antibiotic production and the model parameters were simulated from experimental data by Matlab software. The results demonstrated that the model fitted the experimental data very well.The antifungal compound was identified as a kind of Macrolactins, which not only had inhibitory effect against Aspergillus niger ATCC 20611, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 10031, but also had inhibitory effect against C. albicans ATCC 64548. It possessed great potential of being developed as a new antifungal drug.